Sequences diagnostic for shrimp pathogens

ABSTRACT

Primers have been isolated that are diagnostic for the detection of  Vibrio harveyi . The primers are based on a portion of the  Vibrio harveyi  LuxR gene and may be used in primer directed amplification or nucleic acid hybridization assay methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/873,497, filed Oct. 17, 2007, which claims the benefit of U.S.Provisional Application Ser. No. 60/853,379, filed Oct. 20, 2006, nowexpired, the disclosures of which are herein incorporated by referencein their entirety.

FIELD OF THE INVENTION

The invention relates to the field of diagnostic testing. Morespecifically, new primers have been developed for use in detection ofVibrio harveyi.

BACKGROUND OF THE INVENTION

Commercial shrimp and aquaculture farms suffer extensive losses due tothe effects of a number of common pathogens. Vibrio harveyi, aGram-negative, rod-shaped bacterium, is reported to be the mostimportant bacterial pathogen of the worldwide shrimp aquacultureindustry. Some strains of this bacterium are highly pathogenic toshrimp, while other strains may be considered to be opportunisticpathogens.

Detection of Vibrio harveyi in hatchery broodstock and in post-larvaeallows infected shrimp to be eliminated before entry into a commercialproduction system. Consequently, a variety of methods have beendeveloped for the detection of Vibrio harveyi in shrimp, includingnucleic acid-based methods and immunological methods (Lightner et al.,Aquaculture 164(1):201-220 (1998)). Polymerase chain reaction (PCR)methods are of particular interest because they are simple, rapid, andsensitive. PCR methods for the detection of Vibrio harveyi, which arebased on amplifying different diagnostic regions of the genome, havebeen described (see for example, Conejero et al., J. Gen. Appl.Microbiol. 50(3):137-142 (2004); Conejero et al., J. Appl. Microbiol.95(3):602-611 (2003); and Oakey et al., J. Appl. Microbiol. 95:1293-1303(2003)).

All of the above methods are useful for the detection of Vibrio harveyi;however, they generally suffer from a lack of specificity, sensitivity,or are complex and time consuming. Additionally, because of the highgene mutation rate in the bacterium, tests directed to different regionsof the genome would be useful. Therefore, there is a need for a highlysensitive assay for Vibrio harveyi that is rapid, accurate and easilyused in the field. The stated problem is addressed herein by thediscovery of primers based on a portion of the Vibrio harveyi LuxR genethat may be used in primer directed amplification or nucleic acidhybridization assay methods for the detection of Vibrio harveyi withoutthe problems associated with previous methodologies.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides an isolated Vibrioharveyi diagnostic primer sequence as set forth in SEQ ID NO:1 or SEQ IDNO:2, or an isolated nucleic acid molecule that is complementary to SEQID NO:1 or SEQ ID NO:2.

In another embodiment, the invention provides a pair Vibrio harveyidiagnostic primer sequences as set forth in SEQ ID NO:1 and SEQ ID NO:2.

In another embodiment, the invention provides a kit for the detection ofVibrio harveyi comprising a pair of Vibrio harveyi diagnostic primersequences disclosed herein.

In another embodiment, the invention provides a method for detecting thepresence of Vibrio harveyi in a sample comprising:

-   -   (i) providing DNA from a sample suspected of containing Vibrio        harveyi; and    -   (ii) probing the DNA with a probe derived from the isolated        Vibrio harveyi diagnostic primer sequence of any of SEQ ID        NOs:1-2 under suitable hybridization conditions;    -   wherein the identification of a hybridizable nucleic acid        fragment confirms the presence of Vibrio harveyi.

In another embodiment, the invention provides a method for detecting thepresence of Vibrio harveyi in a sample comprising:

-   -   (i) providing DNA from a sample suspected of containing Vibrio        harveyi; and    -   (ii) amplifying the DNA with a pair of Vibrio harveyi diagnostic        primer sequences disclosed herein such that amplification        products are generated;    -   wherein the presence of amplification products confirms the        presence of Vibrio harveyi.

In another embodiment, the invention provides a method for quantifyingthe amount of Vibrio harveyi in a sample comprising:

-   -   (i) providing DNA from a sample suspected of containing Vibrio        harveyi;    -   (ii) amplifying the DNA with a pair of Vibrio harveyi diagnostic        primer sequences disclosed herein by thermal cycling between at        least a denaturing temperature and an extension temperature in        the presence of a nucleic acid-binding fluorescent agent or a        fluorescently labeled probe;    -   (iii) measuring the amount of fluorescence generated by the        nucleic acid-binding fluorescent agent or the fluorescently        labeled probe during the thermal cycling;    -   (iv) determining a cycle threshold number at which the amount of        fluorescence generated by the nucleic acid-binding fluorescent        agent or the fluorescently labeled probe reaches a fixed        threshold value above a baseline value; and    -   (v) calculating the amount of Vibrio harveyi in the sample by        comparing the cycle threshold number determined for the Vibrio        harveyi in the sample with a standard curve of the cycle        threshold number versus the logarithm of template concentration        determined using standard solutions of known concentration.

BRIEF DESCRIPTION OF THE FIGURE AND SEQUENCE DESCRIPTIONS

The various embodiments of the invention can be more fully understoodfrom the following detailed description, figure, and the accompanyingsequence descriptions, which form a part of this application.

FIG. 1A shows the melting curve for the Vibrio harveyi VHL1 product andthe actin internal sample control product formed by simultaneous PCRamplification of the Vibrio harveyi DNA and actin DNA, as described inExample 5. The melting temperature (Tm) values of the Vibrio harveyi andactin products are indicated on their corresponding melting curves.

FIG. 1B shows the results of an agarose gel electrophoresis separationof samples containing the Vibrio harveyi VH1 product and the actininternal sample control product formed by simultaneous PCR amplificationof the Vibrio harveyi DNA and actin DNA, as described in Example 5. Thequantity of Vibrio harveyi and shrimp DNA is shown above each lane; “M”is a 100-bp DNA ladder.

The following sequences conform with 37 C.F.R. 1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5 (a-bis), and Section 208 and Annex C of theAdministrative Instructions). The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.

SEQ ID NOs:1 and 2 are the nucleotide sequences of Vibrio harveyidiagnostic primers useful for detection of Vibrio harveyi.

SEQ ID NO:3 is the nucleotide sequences of a synthetic Vibrio harveyitemplate described in the General Methods Section of the Examples. Thissequence is also the nucleotide sequence of the amplification productobtained using Vibrio harveyi diagnostic primers, given as SEQ ID NOs:1and 2, in a primer directed amplification reaction.

SEQ ID NOs:4-7 are the nucleotide sequences of internal sample controlprimers described in Example 5.

SEQ ID NOs:8 and 9 are the nucleotide sequences of the fluorescentlylabeled probes described in Examples 6 and 7.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are primers useful in assays for the detection ofVibrio harveyi. The primers may be used in nucleic acid amplificationmethods as well as in hybridization assays for the efficient detectionand quantification of virulent Vibrio harveyi.

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions are provided and should be referred to forinterpretation of the claims and the specification.

“Polymerase chain reaction” is abbreviated PCR.

The term “isolated Vibrio harveyi diagnostic primer sequence” refers toa sequence corresponding to a portion of the Vibrio harveyi genome beingdiagnostic for the presence of Vibrio harveyi.

As used herein, an “isolated nucleic acid fragment” is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid fragment in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA.

The term “amplification product” or “amplicon” refers to the nucleicacid fragment that is produced during a primer directed amplificationreaction. Typical methods of primer directed amplification includepolymerase chain reaction (PCR), ligase chain reaction (LCR), stranddisplacement amplification (SDA) or other isothermal amplificationprocesses. If PCR methodology is selected, the replication compositionwould typically include, for example: deoxynucleotide triphosphates, twoprimers with appropriate sequences, a thermostable DNA polymerase andproteins. These reagents and details describing procedures for their usein amplifying nucleic acids are provided in U.S. Pat. No. 4,683,202(1987, Mullis, et al.) and U.S. Pat. No. 4,683,195 (1986, Mullis, etal.). If LCR methodology is selected, then the nucleic acid replicationcompositions would comprise, for example: a thermostable ligase (e.g.,T. aquaticus ligase), two sets of adjacent oligonucleotides (wherein onemember of each set is complementary to each of the target strands),Tris-HCl buffer, KCl, EDTA, NAD, dithiothreitol and salmon sperm DNA.See, for example, Tabor et al., Proc. Natl. Acad. Sci. U.S.A.,82:1074-1078 (1985)).

The term “primer” refers to an oligonucleotide (synthetic or occurringnaturally), which is capable of acting as a point of initiation ofnucleic acid synthesis or replication along a complementary strand whenplaced under conditions in which synthesis of a complementary stand iscatalyzed by a polymerase.

The term “thermal cycling” refers to the entire pattern of changingtemperature used during certain nucleic acid amplification methods, suchas PCR and LCR. This process is common and well known in the art. See,for example, Sambrook, J., Fritsch, E. F. and Maniatis, T., MolecularCloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor LaboratoryPress: Cold Spring Harbor, N.Y. (1989); and U.S. Pat. No. 4,683,202 toMullis et al. and U.S. Pat. No. 4,683,195 to Mullis et al. In general,PCR thermal cycling includes an initial denaturing step at hightemperature, followed by a repetitive series of temperature cyclesdesigned to allow template denaturation, primer annealing, and extensionof the annealed primers by the polymerase.

The term “cycle threshold number”, also referred to herein as “CT”,refers to the cycle number during thermal cycling at which the amount offluorescence due to product formation reaches a fixed threshold valueabove a baseline value.

The term “probe” refers to an oligonucleotide (synthetic or occurringnaturally) that is significantly complementary to a target sequence,also referred to herein as a “fragment”, (i.e., the sequence to bedetected or a portion of the sequence to be detected) and forms aduplexed structure by hybridization with at least one strand of thetarget sequence. The probe can be labeled to facilitate detection, forexample, using a fluorescent label or a ligand label.

The term “replication inhibitor moiety” refers to any atom, molecule orchemical group that is attached to the 3′ terminal hydroxyl group of anoligonucleotide that will block the initiation of chain extension forreplication of a nucleic acid strand. Examples include, but are notlimited to, 3′ deoxynucleotides (e.g., cordycepin), dideoxynucleotides,phosphate, ligands (e.g., biotin and dinitrophenol), reporter molecules(e.g., fluorescein and rhodamine), carbon chains (e.g., propanol), amismatched nucleotide or polynucleotide, or peptide nucleic acid units.

The term “non-participatory” refers to the lack of participation of aprobe or primer in a reaction for the amplification of a nucleic acidmolecule. Specifically, a non-participatory probe or primer is one thatwill not serve as a substrate for, or be extended by, a DNA polymerase.A “non-participatory probe” is inherently incapable of being chainextended by a polymerase. It may or may not have a replication inhibitormoiety.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under suitable conditions of temperature and solution ionicstrength. Hybridization and washing conditions are well known andexemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor LaboratoryPress: Cold Spring Harbor, N.Y. (1989), particularly Chapter 11 andTable 11.1 therein (entirely incorporated herein by reference). Theconditions of temperature and ionic strength determine the “stringency”of the hybridization. For preliminary screening for homologous nucleicacids, low stringency hybridization conditions, corresponding to amelting temperature (Tm) of 55° C., can be used, e.g., 5×SSC, 0.1% SDS,0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5% SDS.Moderate stringency hybridization conditions correspond to a higher Tm,e.g., 40% formamide, with 5× or 6×SSC. Hybridization requires that thetwo nucleic acids contain complementary sequences, although depending onthe stringency of the hybridization, mismatches between bases arepossible. The appropriate stringency for hybridizing nucleic acidsdepends on the length of the nucleic acids and the degree ofcomplementation, variables well known in the art. The greater the degreeof similarity or homology between two nucleotide sequences, the greaterthe value of Tm for hybrids of nucleic acids having those sequences. Therelative stability (corresponding to higher Tm) of nucleic acidhybridizations decreases in the following order: RNA:RNA, DNA:RNA,DNA:DNA. For hybrids of greater than 100 nucleotides in length,equations for calculating Tm have been derived (see Sambrook et al.,supra, 9.50-9.51). For hybridizations with shorter nucleic acids, i.e.,oligonucleotides, the position of mismatches becomes more important, andthe length of the oligonucleotide determines its specificity (seeSambrook et al., supra, 11.7-11.8). In one embodiment, the length for ahybridizable nucleic acid is at least about 10 nucleotides. Preferably,a minimum length for a hybridizable nucleic acid is at least about 15nucleotides; more preferably at least about 20 nucleotides; and mostpreferably the length is at least 30 nucleotides. Furthermore, theskilled artisan will recognize that the temperature and wash solutionsalt concentration may be adjusted as necessary according to factorssuch as length of the probe.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide.

The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include, but is not limited to, the GCG suite of programs(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.215:403-410 (1990), DNASTAR (DNASTAR, Inc., Madison, Wis.), and VectorNTI® software version 7.0. Within the context of this application itwill be understood that where sequence analysis software is used foranalysis, that the results of the analysis will be based on the “defaultvalues” of the program referenced, unless otherwise specified. As usedherein “default values” will mean any set of values or parameters whichoriginally load with the software when first initialized.

Standard recombinant DNA and molecular cloning techniques used here arewell known in the art and are described by Sambrook, J., Fritsch, E. F.and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2^(nd) ed.,Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. (1989)(hereinafter “Maniatis”); and by Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, published by Greene Publishing Assoc.and Wiley-Interscience (1987).

Vibrio harveyi Genome

Vibrio harveyi is a major bacterial shrimp pathogen with a highmortality rate and a wide host range. The complete genome of Vibrioharveyi has not been sequenced.

Vibrio harveyi Diagnostic Primer Sequences

Disclosed herein are diagnostic primer sequences useful in a variety ofassay formats for high sensitive detection of Vibrio harveyi. Theseprimers are directed to regions of the Vibrio harveyi LuxR gene notpreviously used for Vibrio harveyi detection. (Showalter et al., J.Bacteriol. 172(6):2946-2954 (1990), GenBank: M55260).

Primer sequences were empirically identified using a series of “insilica” (i.e. computer-based) sequence analysis tools. In this process,a database was assembled containing all known Vibrio harveyi genomesequences. These sequences were first aligned and then analyzed forprimer sites using Vector NTI® software (InforMax Inc., Bethesda, Md.)based on homology with other Vibrio harveyi genome sequences, aspecified amplicon length, salt concentration, Tm (melting temperature),C+G content and freedom from hairpin and secondary structure parameters.Prospective primers were then screened against GenBank sequences. Thoseprimers established to contain less than 5 bases of homology with othernon-target gene sequences were selected for experimental investigationof PCR amplification efficiency and minimal primer-dimer formation.Primers showing both high amplification efficiency and minimalprimer-dimer formation were selected for testing with a panel of DNAisolated from shrimp infected with various shrimp pathogens and DNA fromshrimp certified to be disease free. Those primers amplifying all Vibrioharveyi strains and showing no response to both DNA from shrimp infectedwith non-Vibrio harveyi pathogens and to DNA isolated from differentspecies of certified disease free shrimp were selected as usefulprimers.

The primer sequences found to be useful in the detection of Vibrioharveyi and their location in the Vibrio harveyi LuxR gene are given inTable 1. These primers may be synthesized using standard phosphoramiditechemistry or may be purchased from companies such as Sigma Genosys (TheWoodlands, Tex.).

TABLE 1 Vibrio harveyi Diagnostic Primer Sequences SEQ Location inVibrio harveyi LuxR Primer, Direction ID NO: Gene (GenBank M55260)VHL1F, Forward 1 679-700 VHL1R, Reverse 2 758-737

Assay Methods

The primer sequences disclosed herein may be used in a variety of assayformats for the detection and quantification of Vibrio harveyi. The twomost convenient formats rely on methods of nucleic acid hybridization orprimer directed amplification methods such as PCR.

Primer Directed Amplification Assay Methods

In one embodiment, the present Vibrio harveyi diagnostic primersequences may be used in primer directed nucleic acid amplification forthe detection of the presence of Vibrio harveyi. A variety of primerdirected nucleic acid amplification methods are well known in the artand are suitable for use with the primers disclosed herein. Thesenucleic acid amplification methods include thermal cycling methods(e.g., polymerase chain reaction (PCR) and ligase chain reaction (LCR)),as well as isothermal methods and strand displacement amplification(SDA).

LCR methods are well known in the art (see for example, Tabor et al.,Proc. Natl. Acad. Sci. U.S.A., 82:1074-1078 (1985)). Typically, LCRnucleic acid replication compositions comprise, for example: athermostable ligase (e.g., T. aquaticus ligase), two sets of adjacentoligonucleotide primers (wherein one member of each set is complementaryto each of the target strands), Tris-HCl buffer, KCl, EDTA, NAD,dithiothreitol and salmon sperm DNA.

SDA methods are also well known in the art. An in depth discussion ofSDA methodology is given by Walker et al. (Proc. Natl. Acad. Sci.U.S.A., 89:392 (1992)). Typically in SDA, two oligonucleotide primersare used, each having regions complementary to only one of the stands inthe target. After heat denaturation, the single-stranded targetfragments bind to the respective primers which are present in excess.Both primers contain asymmetric restriction enzyme recognition sequenceslocated 5′ to the target binding sequences. Each primer-target complexcycles through nicking and polymerization/displacement steps in thepresence of a restriction enzyme, a DNA polymerase and threedeoxynucleotide triphosphates (dNTPs) and one deoxynucleotide α-thiotriphosphate (dNTP[aS]).

The preferred method for detecting Vibrio harveyi using the diagnosticprimer sequences disclosed herein is PCR, which is described by Mulliset al. in U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,683,195, which areboth specifically incorporated herein by reference. In PCR methods theVibrio harveyi diagnostic primer sequences disclosed herein are used asa pair which is capable of priming a nucleic acid amplification reactionthat amplifies a region within the Vibrio harveyi genome. Specifically,the VHL1F forward primer, given as SEQ ID NO:1, is used with the VHL1Rreverse primer given as SEQ ID NO:2. Generally, the two primers aremixed with the sample DNA, a mixture of four deoxynucleotidetriphosphates (i.e., dATP, dCTP, dTTP, and dGTP), a thermostable DNApolymerase, such as Taq DNA polymerase, in a buffer solution. Thismixture is then thermal cycled using a thermal cycler instrument toamplify the desired target region. Thermal cyclers are commerciallyavailable from many sources (e.g., Applied Biosystems (Foster City,Calif.); Brinkmann (Westbury, N.Y.); MJ Research (Waltham, Mass.); andStratagene (La Jolla, Calif.)).

In general, PCR thermal cycling includes an initial denaturing step athigh temperature, followed by a repetitive series of temperature cyclesdesigned to allow template denaturation, primer annealing, and extensionof the annealed primers by the polymerase. Generally, the samples areheated initially for about 2 to 10 minutes at a temperature of about 95°C. to denature the double stranded DNA sample. Then, in the beginning ofeach cycle, the samples are denatured for about 10 to 60 seconds,depending on the samples and the type of instrument used. Afterdenaturing, the primers are allowed to anneal to the target DNA at alower temperature, from about 40° C. to about 60° C. for about 20 to 60seconds. Extension of the primers by the polymerase is often carried outat a temperature ranging from about 60° C. to about 72° C. The amount oftime used for extension will depend on the size of the amplicon and thetype of enzymes used for amplification and is readily determined byroutine experimentation. Additionally, the annealing step can becombined with the extension step, resulting in a two step cycling.Thermal cycling may also include additional temperature shifts in PCRassays. The number of cycles used in the assay depends on many factors,including the primers used, the amount of sample DNA present, and thethermal cycling conditions. The number of cycles to be used in any assaymay be readily determined by one skilled in the art using routineexperimentation. Optionally, a final extension step may be added afterthe completion of thermal cycling to ensure synthesis of allamplification products.

Following amplification, the amplified nucleotide sequence may beligated to a suitable vector followed by transformation of a suitablehost organism with said vector. One thereby ensures a more readilyavailable supply of the amplified sequence. Alternatively, followingamplification, the amplified sequence or a portion thereof may bechemically synthesized for use as a nucleotide probe for use in ahybridization assay, as described below. In either situation the DNAsequence of the variable region may be established using methods such asthe dideoxy method (Sanger, F. et al. Proc. Natl. Acad. Sci.74:5463-5467 (1977)). The sequence obtained is used to guide the choiceof the probe for the organism and the most appropriate sequence(s)is/are selected.

In order to detect the presence of Vibrio harveyi in a sample suspectedof containing Vibrio harveyi (e.g., shrimp or other crustaceans) using aprimer directed nucleic acid amplification method, DNA from the samplemust be provided in a form that is capable of being amplified.

Typically, the DNA must be free from the cell and sample materials andmay be treated to eliminate proteins and other cell components. The DNAmay be obtained from bacteria from any suitable tissue, fluid or samplematerial including, but not limited to, shrimp tissue (gills, pleopods,hemolymph, muscle, tail, eyestalk, stomach, leg, and connective tissue),wash fluids, and pond water samples. Typically, bacterial cells areisolated from sample materials. These samples can be cultured in asuitable growth medium, such as tryptic soy broth supplemented withNaCl, blood agar supplemented with NaCl, Marine broth, or thiosulfatecitrate bile sucrose (TCBS) broth. Alternatively, DNA can be extracteddirectly from the sample for testing. The samples may be suspected ofcontaining Vibrio harveyi for any number of reasons, including proximityto a known contaminant or otherwise, or may only be suspected ofcontamination by virtue of Vibrio harveys common presence in thecommercial shrimp industry. Thus, a sample suspected of containingVibrio harveyi can be any DNA sample described above.

Methods for providing DNA, which is suitable for amplification, fromsamples are well known in the art (Maniatis, supra). The DNA can beextracted directly from the sample material. Alternatively, the DNA canbe extracted from a bacterial sample, for example, by harvesting thecells by centrifugation, lysing the cells with sodium dodecylsulfate(SDS) and, extracting the DNA with phenol and chloroform-isoamylalcohol, as described by Goarant et al. (Appl. Environ. Microbiol.65(3):1145-1151 (1999)). Additionally, the DNA can be provided in a formwhich is suitable for amplification using a commercially available DNAisolation kit, such as the QIAamp DNA Mini Kit (Qiagen, ValenciaCalif.), the QIAamp Tissue Kit (Qiagen), or the High Pure PCR TemplatePreparation Kit (Roche Applied Science, Indianapolis, Ind.).

The DNA is then amplified with the pair of diagnostic primer sequencesdisclosed herein using a nucleic acid amplification method, as describedabove. The presence of the amplification product, detected as describedbelow, confirms the presence of Vibrio harveyi in the sample. In oneembodiment, PCR is used to amplify the DNA.

In nucleic acid amplification methods, test results can bemisinterpreted due to reagent failure, procedural errors, and instrumentmalfunction. Additionally, problems arise due to the presence ofinhibitory substances in the sample materials or degradation of thesample DNA or RNA during sample processing and nucleic acid recovery. Toovercome these problems, internal control tests can be performed incombination with the Vibrio harveyi assay to alert users to these typesof errors and to aid in quantification of test results.

Two types of internal control tests can be used. One approach is basedon co-amplification of an “internal template control” (ITC), which isadded to the nucleic acid amplification reagent mixture prior toreaction. A second approach is based on co-amplification of an “internalsample control” (ISC) contained in the sample. In both cases, thesequence of the internal control DNA or RNA is different from that ofthe Vibrio harveyi DNA.

The internal sample control can be a DNA or RNA gene sequence conservedor consistently present in sample materials (e.g., shrimp tissue andhemolymph). The primers used to amplify the ISC target DNA or RNA arechosen so that they do not amplify Vibrio harveyi DNA and the Vibrioharveyi test primers are chosen so that they do not amplify the internalsample control DNA or RNA targets. In this way, the ISC and Vibrioharveyi targets amplify independently. In the assay, both the ISC andthe Vibrio harveyi targets are processed using the same reagents andconditions. Furthermore, both target templates are amplified using thesame reagents and reaction conditions. Because the ISC template andprimers are present in the test samples, ISC product should be producedduring amplification. If the ISC product is not formed, it is anindication that the test chemistry did not function correctly and theVibrio harveyi test results are incorrect and should not be relied on.The formation of the correct ISC indicates that the test chemistryworked correctly, and the Vibrio harveyi sample processing and testreactions are assumed to have functioned correctly. In these instances,the Vibrio harveyi test can be more accurately interpreted.

ISC primers can be selected from gene sequences of genes coding forstructural proteins, metabolic enzymes or ribosomal products of thepathogen host species which are subject to Vibrio harveyi infections.For example, the ISC primers can be gene sequences derived from theshrimp actin gene, or 18S, 23S or 5S ribosomal genes of shrimp, or otherconstitutive genes in test organisms. Suitable examples of ISC primerpairs include, but are not limited to, SEQ ID NOs: 4 and 5, and SEQ IDNOs: 6 and 7, derived from the Penaeus monodon actin 1 gene (GenBankAF100986), as shown in Table 2.

TABLE 2 Internal Sample Control (ISC) Primer Sequences Actin 1 GeneLocation Primer, Direction SEQ ID NO: (GenBank AF100986) ActinF2,Forward 4 391-411 ActinR2, Reverse 5 608-629 ActinF3, Forward 6 326-346ActinR3, Reverse 7 553-574

In one embodiment, at least one pair of ISC primers is included in thenucleic acid amplification reagent mixture in order to produce aninternal sample control product in the amplification reaction. In oneembodiment, the at least one pair of ISC primers is selected from thegroup consisting of SEQ ID NOs:4 and 5, and SEQ ID NOs:6 and 7.

Additionally, an internal template control (ITC) can be used toadvantage with the Vibrio harveyi test primers to aid in quantificationof the test response. Primer requirements for the ITC are similar tothose of the ISC primers with the exception that both the ITC templateand primers are added to the amplification reagent mixture. The ITCprimers are chosen so that they do not amplify genomic DNA or RNA fromthe test species, such as shrimp, which are subject to Vibrio harveyiinfection. The ITC template is added at a known concentration so thatthe copy number per reaction is known. Because the ITC template isincluded in the amplification reagent mixture, the ITC product isproduced during amplification. The amount of ITC product will vary fromreaction to reaction depending on the amplification efficiency of thereaction and other variables. Since these same variables also affect theVibrio harveyi DNA amplification, the amount of Vibrio harveyi productproduced will be proportionately related to the amount of the ITCproduct produced in the reaction. Therefore, the copy number of theVibrio harveyi template in the assay can be inferred from theproportionality between the ITC originally added, the ITC productformed, and the Vibrio harveyi product produced. Relative productformation can be determined in CT units when labeled internal probes areused or by the derivative of the melting curves at the products'respective melt temperature.

The ITC primer sequences can be rationally designed or derived from genesequences from non-test species such as other viruses or genes fromplants and animals which are not present in the test samples. In thisway, sample materials do not contain other DNA or RNA which could beamplified by the ITC primers.

In one embodiment, at least one internal template control and at leastone pair of ITC primers are included in the nucleic acid amplificationreagent mixture in order to produce at least one ITC product in theamplification reaction.

A variety of detection methods, which are well known in the art, may beused in the methods disclosed herein. These detection methods include,but are not limited to, standard non-denaturing gel electrophoresis(e.g., acrylamide or agarose), denaturing gradient gel electrophoresis,temperature gradient gel electrophoresis, capillary electrophoresis, andfluorescence detection.

Fluorescence detection methods provide rapid and sensitive detection ofamplification products. Fluorescence detection also provides thecapability of real-time detection, wherein the formation ofamplification products is monitored during the thermal cycling process.Additionally, the amount of the initial target may be quantified usingfluorescence detection. Fluorescence detection may be done by adding anucleic acid-binding fluorescent agent to the reaction mixture eitherbefore or after the thermal cycling process. Preferably, the nucleicacid-binding fluorescent agent is an intercalating dye that is capableof non-covalent insertion between stacked base pairs in the nucleic aciddouble helix. However, non-intercalating nucleic acid-bindingfluorescent agents are also suitable. Non-limiting examples of nucleicacid-binding fluorescent agents useful in the methods of the inventionare ethidium bromide and SYBR® Green I (available from Molecular Probes;Eugene, Oreg.). Addition of the nucleic acid-binding fluorescent agentto the reaction mixture prior to thermal cycling permits monitoring ofthe formation of amplification products in real-time, as described byHiguchi (U.S. Pat. No. 5,994,056). Thermal cyclers capable of real-timefluorescence measurements are commercially available from companies suchas Applied Biosystems (Foster City, Calif.), MJ Research (Waltham,Mass.), and Stratagene (La Jolla, Calif.). Following amplification,confirmation of the amplification product can be assessed by determiningthe melting temperature of the product using methods know in the art,for example, by generating a melting curve using fluorescencemeasurement.

Fluorescence detection of amplification products may also beaccomplished using other methods known in the art, such as the use of afluorescently labeled probe. The probe comprises a complementarysequence to at least a portion of the amplification product.Non-limiting examples of such probes include TaqMan® probes (AppliedBiosystems) and Molecular Beacons (Goel et al., J. Appl. Microbiol.99(3):435-442 (2005)). For example, gene sequences for the constructionof fluorescently labeled probes for use with the Vibrio harveyi primersdisclosed herein can be selected by analysis of the Vibrio harveyi genesand test amplicons using commercially available software such as PrimerExpress® v2.0 (Applied BioSystems Inc., Foster City Calif.), asdescribed in Examples 6 and 7. Probe sequences are selected to fallwithin the proximal ends of the specific Vibrio harveyi test amplicons.Suitable probe sequences include, but are not limited, to the sequencesset forth in SEQ ID NOs:8 and 9. The probes may be fluorescently labeledusing methods known in the art, such as those described below forlabeling hybridization probes. For real time fluorescent detection,probes can be dual labeled. For example, the 5′ end of the probe can belabeled with a fluorophore, such as 6FAM™ (Applied BioSystems), and the3′ end can be labeled with a quencher dye, such as6-carboxytetramethylrhodamine (TAMRA). In the case of a minor groovebinding probe, the 3′ end can be labeled with a quencher dye and a minorgroove binder complex. Fluorescently labeled probes may be obtained fromcommercial sources such as Applied BioSystems.

In one embodiment, the invention provides a method for quantifying theamount of Vibrio harveyi in a sample. In this embodiment, DNA isprovided from a sample suspected of containing Vibrio harveyi, asdescribed above. The DNA is amplified with the pair of theoligonucleotide primers disclosed herein by thermal cycling between atleast a denaturing temperature and an extension temperature in thepresence of a nucleic acid-binding fluorescent agent or a fluorescentlylabeled probe. The amount of fluorescence generated by the nucleicacid-binding fluorescent agent or the fluorescently labeled probe ismeasured during thermal cycling. From the fluorescence measurements, acycle threshold number is determined at which the amount of fluorescencegenerated by the nucleic acid-binding fluorescent agent or thefluorescently labeled probe reaches a fixed threshold value above abaseline value. The cycle threshold number is referred to herein as theCT number or value. The CT number can be determined manually ordetermined automatically by the instrument. To determine the CT number,the baseline fluorescence is determined for each sample during theinitial amplification cycles. A mathematical algorithm is then employedto establish what a statistically significant change in fluorescencewould need to be for the fluorescence signal to be above the background.The cycle number at which the florescence exceeds this threshold isreferred to as the CT number. Typically, the more DNA present in thesample at the start of the thermal cycling, the fewer number of cyclesit will take to reach the threshold value. Therefore, the CT number isinversely related to the initial amount of Vibrio harveyi in the sample.After the CT number for the Vibrio harveyi sample is determined, theamount of Vibrio harveyi originally present in the sample can becalculated by comparing the cycle threshold number determined for theVibrio harveyi in the sample with a standard curve of the cyclethreshold number versus the logarithm of template concentrationdetermined using standard solutions of known concentration, as is wellknown in the art.

Nucleic Acid Hybridization Methods

The basic components of a nucleic acid hybridization test for Vibrioharveyi include a DNA probe, a sample suspected of containing Vibrioharveyi, and a specific hybridization method. Probes of the presentinvention are single stranded nucleic acid sequences which arecomplementary to the nucleic acid sequences to be detected and are“hybridizable” thereto. Typically in hybridization methods, the probelength can vary from as few as 5 bases to several kilobases and willdepend upon the specific test to be done. Only part of the probemolecule need be complementary to the nucleic acid sequence to bedetected. In addition, the complementarity between the probe and thetarget sequence need not be perfect. Hybridization does occur betweenimperfectly complementary molecules with the result that a certainfraction of the bases in the hybridized region are not paired with theproper complementary base.

The DNA probes disclosed herein are derived from the Vibrio harveyidiagnostic primer sequences described above. As used herein the phrase“derived from the Vibrio harveyi diagnostic primer sequences” means thatthe DNA probes can be the Vibrio harveyi diagnostic primer sequences,the amplification product sequences obtained therefrom using a nucleicacid amplification method, portions of the Vibrio harveyi diagnosticprimer sequences or the amplification product sequences, or the completecomplementary sequences of any of the aforementioned sequences. The term“portion”, as used above, refers to any part of the Vibrio harveyidiagnostic primer sequences or the amplification products obtainedtherefrom that is less than the complete sequence. Preferably, thelength of the portion for use as a probe is at least about 15 bases,more preferably, at least about 20 bases. Non-limiting examples of DNAprobes derived from the WSSV diagnostic primer sequences include theVibrio harveyi diagnostic primer sequences given as SEQ ID NOs:1 and 2,the amplification product sequence given as SEQ ID NO:3, and thecomplete complementary sequences thereof.

The probe may be labeled to facilitate detection. Methods of attachinglabels to nucleic acid probes are well known in the art. For example,the probe can be labeled during synthesis by incorporation of labelednucleotides. Alternatively, probe labeling can be done by nicktranslation or end-labeling. The label may comprise a fluorophore forfluorescence detection, or a ligand, such as biotin, which is detectedusing an enzyme-labeled binding molecule that binds to the ligand (e.g.,enzyme-labeled streptavidin) subsequent to hybridization.

In order to detect the presence of Vibrio harveyi in a sample suspectedof containing Vibrio harveyi, DNA is provided from the sample, asdescribed above. The sample DNA is made available to contact the probebefore any hybridization of probe and target molecule can occur. Thus,the DNA must be free from the cell and placed under the properconditions before hybridization can occur. Additionally in someembodiments, it may be desirable to purify the DNA to eliminateproteins, lipids, and other cell components. A variety of methods ofnucleic acid purification, such as phenol-chloroform extraction, areknown to those skilled in the art (Maniatis, supra). Additionally, kitsare available from commercial sources for DNA extraction andpurification, as described above. Pre-hybridization purification isparticularly useful for standard filter hybridization assays.

In one embodiment, hybridization assays may be conducted directly oncell lysates, without the need to extract the nucleic acids. Thiseliminates several steps from the sample-handling process and speeds upthe assay. To perform such assays on crude cell lysates, a chaotropicagent is typically added to the cell lysates prepared as describedabove. The chaotropic agent stabilizes nucleic acids by inhibitingnuclease activity. Furthermore, the chaotropic agent allows sensitiveand stringent hybridization of short oligonucleotide probes to DNA atroom temperature (Van Ness and Chen, Nucl. Acids Res. 19:5143-5151(1991)). Suitable chaotropic agents include guanidinium chloride,guanidinium thiocyanate, sodium thiocyanate, lithium tetrachloroacetate,sodium perchlorate, rubidium tetrachloroacetate, potassium iodide, andcesium trifluoroacetate, among others. Typically, the chaotropic agentis present at a final concentration of about 3 M. If desired, one canadd formamide to the hybridization mixture, typically 30 to 50% byvolume.

Hybridization methods are well defined and include solution (i.e.,homogeneous) and solid phase (i.e., heterogeneous) hybridizationmethods. Typically, the sample DNA is probed (i.e. contacted underconditions which will permit nucleic acid hybridization) with a probederived from the Vibrio harveyi diagnostic primer sequences disclosedherein. This involves contacting the probe and sample DNA in thepresence of an inorganic or organic salt under the proper concentrationand temperature conditions. The probe and sample nucleic acids must bein contact for a long enough time such that any possible hybridizationbetween the probe and sample nucleic acid may occur. The concentrationof probe or target in the mixture will determine the time necessary forhybridization to occur. The higher the probe or target concentration,the shorter the hybridization incubation time needed.

Various hybridization solutions can be employed. Typically, these maycomprise from about 20 to 60% by volume, preferably 30%, of a polarorganic solvent. A common hybridization solution employs about 30 to 50%by volume formamide, about 0.15 to 1 M sodium chloride, about 0.05 to0.1 M buffers, such as sodium citrate, Tris-HCl, PIPES or HEPES (pHrange about 6-9), about 0.05 to 0.2% detergent, such as sodiumdodecylsulfate (SDS), between 0.5 to 20 mM EDTA, FICOLL (AmershamBioscience Inc., Piscataway, N.J.) (molecular weight of about 300-500kilodaltons), polyvinylpyrrolidone (molecular weight of about 250-500kilodaltons), and serum albumin. Also included in a typicalhybridization solution may be unlabeled carrier nucleic acids from about0.1 to 5 mg/mL, fragmented nucleic DNA (e.g., calf thymus or salmonsperm DNA, or yeast RNA), and optionally from about 0.5 to 2% weight pervolume glycine. Other additives may also be included, such as volumeexclusion agents which include a variety of polar water-soluble orswellable agents (e.g., polyethylene glycol), anionic polymers (e.g.,polyacrylate or polymethylacrylate), and anionic saccharidic polymers(e.g., dextran sulfate).

Nucleic acid hybridization is adaptable to a variety of assay formats.One of the most suitable is the sandwich assay format. The sandwichassay is particularly adaptable to hybridization under non-denaturingconditions. A primary component of a sandwich-type assay is a solidsupport. The solid support has adsorbed to it or covalently coupled toit, an immobilized nucleic acid capture probe that is unlabeled and iscomplementary to one portion of the sample DNA sequence. Probesparticularly useful in the present invention are those derived from thepresent Vibrio harveyi diagnostic sequences, as described above. Thecaptured DNA is detected using a second probe that is labeled, asdescribed above, and is complementary to a different portion of thesample DNA sequence. The label may be detected using methods known inthe art (e.g., fluoresence, chemiluminescence, binding pair enzyme assayand the like).

Hybridization methods may also be used in combination with nucleic acidamplification methods, such as PCR. For example, the instant Vibrioharveyi diagnostic sequences may be used as 3′ blocked detection probesin either a homogeneous or heterogeneous assay format. For example, aprobe generated from the instant sequences may be 3′ blocked ornon-participatory and will not be extended by, or participate in, anucleic acid amplification reaction. Additionally, the probeincorporates a label that can serve as a reactive ligand that acts as apoint of attachment for the immobilization of the probe/analyte hybridor as a reporter to produce detectable signal. Accordingly, genomic DNAisolated from a sample suspected of harboring Vibrio harveyi isamplified by standard primer-directed amplification protocols in thepresence of an excess of the 3′ blocked detection probe to produceamplification products. Because the probe is 3′ blocked, it does notparticipate or interfere with the amplification of the target. After thefinal amplification cycle, the detection probe anneals to the relevantportion of the amplified DNA and the annealed complex is then capturedon a support through the reactive ligand.

The instant probe is versatile and may be designed in several alternateforms. The 3′ end of the probe may be blocked from participating in aprimer extension reaction by the incorporation or attachment of areplication inhibiting moiety. Typical replication inhibitor moietiesinclude, but are not limited to, dideoxynucleotides, 3′deoxynucleotides, a sequence of mismatched nucleosides or nucleotides,3′ phosphate groups and chemical agents, such as biotin, dinitrophenol,fluorescein, rhodamine, and carbon chains. The replication inhibitor iscovalently attached to the 3′ hydroxy group of the 3′ terminalnucleotide of the non-participatory probe during chemical synthesis,using standard cyanoethyl phosphoramidite chemistry. This process usessolid phase synthesis chemistry in which the 3′ end is covalentlyattached to an insoluble support (controlled pore glass, or “CPG”) whilethe newly synthesized chain grows on the 5′ terminus. Within the contextof the present invention, 3-deoxyribonucleotides are the preferredreplication inhibitors. Cordycepin (3-deoxyadenosine) is most preferred.Since the cordycepin will be attached to the 3′ terminal end of theprobe sequence, the synthesis is initiated from a cordycepin covalentlyattached to CPG, 5-dimethoxytrityl-N-benzoyl-3-deoxyadenosine(cordycepin), 2-succinoyl-long chain alkylamino-CPG (Glen Research,Sterling, Va.). The dimethoxytrityl group is removed and the initiationof the chain synthesis starts at the deprotected 5′ hydroxyl group ofthe solid phase cordycepin. After the synthesis is complete, theoligonucleotide probe is cleaved off the solid support leaving a free 2′hydroxyl group on the 3′-terminally attached cordycepin. Other reagentscan also be attached to the 3′ terminus during the synthesis of thenon-participatory probe to serve as replication inhibitors.

These include, but are not limited to, other 3-deoxyribonucleotides,biotin, dinitrophenol, fluorescein, and digoxigenin. CPG supports,derivatized with each of these reagents, are available from commercialsources (e.g., Glen Research, Sterling, Va.; and CLONTECH Laboratories,Palo Alto, Calif.).

Alternatively, asymmetric amplification may be used to generate a strandcomplementary to the detection probe. Asymmetric PCR conditions forproducing single-stranded DNA are similar to the conditions describedabove for PCR; however, the primer concentrations are adjusted so thatone primer is in excess and the other primer is limiting. It iscontemplated that this procedure would increase the sensitivity of themethod. This improvement in sensitivity would occur by increasing thenumber of available single strands for binding with the detection probe.

Assessment Vibrio harveyi Inactivation

The methods for detecting the presence of and quantifying the amount ofVibrio harveyi in a sample disclosed herein may be used to assess theextent of Vibrio harveyi inactivation. For example, the methodsdisclosed herein may be used in combination with a chemical treatment toimprove the health and grow-out of shrimp. Specifically, duringproduction and grow-out, the shrimp, samples taken from the productionfacilities, or samples taken from the shrimp's environment may besampled and tested for the presence of Vibrio harveyi using the methodsdisclosed herein. If Vibrio harveyi is found, the facilities and/or theshrimp can be treated to kill or control the bacterium. Because of thehigh sensitivity of the test, Vibrio harveyi can be detected early,before devastation and loss of the crop. Thus, use of the methodsdisclosed herein in combination with chemical intervention can improveproduction efficiency and yield. Examples of chemical treatmentsinclude, but are not limited to, oxidative disinfectants such as Virkon®S disinfectant (a registered trademark of E.I. Du Pont de Nemours andCo.), peracetic acids, hydrogen peroxide, permanganate, potassiummonopersulfate, hypochlorous acid, hypochlorite, iodine and the like;probiotics, immunostimulants, and feed supplements. After the chemicaltreatment, the shrimp can be sampled and retested to determine if thetreatment was successful in eradicating the bacterium.

Detection Kits

In another embodiment, the invention provides a kit for the detection ofVibrio harveyi based on a nucleic acid amplification method. The kitcomprises a pair of Vibrio harveyi diagnostic primer sequences, asdescribed above. Additionally, the kit may further comprise at least oneof the following reagents: a thermostable DNA polymerase, a mixture offour different deoxynucleotide triphosphates, a nucleic acid-bindingfluorescence agent, at least one pair of internal sample controlprimers, at least one internal template control and at least one pair ofinternal template control primers, a probe comprising a complementarysequence to a portion of at least one region of nucleic acid within theVibrio harveyi genome which is capable of being amplified with theVibrio harveyi diagnostic primer sequences contained in the kit. Theprimers and other reagents of the kit may be in various forms, such as aliquid, dried, or tablet and may be present in any suitable container ormultiple containers, such as vials, tubes, and the like.

In another embodiment, the invention provides a kit for the detection ofVibrio harveyi based on a sandwich assay hybridization method. This kitcomprises a first component for the collection of samples from a shrimpor other crustacean suspected of having contracted Vibrio harveyi andbuffers for the disbursement and lysis of the sample. A second componentincludes media in either dry or liquid form for the hybridization oftarget and probe nucleic acids, as well as for the removal ofundesirable and non-hybridized forms by washing. A third componentincludes a solid support (e.g., dipstick, bead, and the like) upon whichis fixed (or to which is conjugated) unlabeled nucleic acid probe(s)that is (are) derived from the isolated Vibrio harveyi diagnostic primersequences disclosed herein. A fourth component contains labeled probethat is complementary to a second and different region of the same DNAstrand to which the immobilized, unlabeled nucleic acid probe of thethird component is hybridized. The labeled probe may also be derivedfrom the isolated Vibrio harveyi diagnostic primer sequences disclosedherein.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various uses andconditions.

The meaning of abbreviations is as follows: “sec” means second(s), “min”means minute(s), “hr” means hour(s), “d” means day(s), “μL” meansmicroliter(s), “mL” means milliliter(s), “L” means liter(s), “μM” meansmicromolar, “mM” means millimolar, “nM” means nanomolar, “M” meansmolar, “mmol” means millimole(s), “μmol” mean micromole(s), “ng” meansnanogram(s), “fg” means femtogram(s), “μg” means microgram(s), “mg”means milligram(s), “g” means gram(s), “nm” means nanometer(s), “mU”means milliunit(s), “U” means unit(s), “rxn” means reaction(s), “PCR”means polymerase chain reaction, “OD” means optical density, “OD₂₆₀”means the optical density measured at a wavelength of 260 nm, “OD₂₈₀”means the optical density measured at a wavelength of 280 nm,“OD_(280/260)” means the ratio of the OD₂₈₀ value to the OD₂₆₀ value,“rpm” means revolutions per minute, “CT” means the cycle number at whichthe buildup in fluorescence in the reaction exceeds the detectionthreshold, and “SPF” means certified specific pathogen free.

General Methods

Standard recombinant DNA and molecular cloning techniques used in theExamples are well known in the art and are described by Sambrook, J.,Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, byT. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with GeneFusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1984, and by Ausubel, F. M. et al., Current Protocols in MolecularBiology, Greene Publishing Assoc. and Wiley-Interscience, N.Y., 1987.

Analysis of genome sequences and primer designates was accomplishedusing the Vector NTI® Software Suite available from InforMax Inc.(Bethesda, Md.).

Enzymes and reagents used herein were purchased from the followingvendors:

-   -   Applied Biosystems, Foster City, Calif.: AmpliTaq (Catalog No.        N808-0160);    -   New England Biolabs, Beverly, Mass.: deoxynucleotide solution        mix (Catalog No. N0447S);    -   Sigma Genosys, The Woodlands, Tex.: Oligonucleotides; Invitrogen        Life Technologies, Carlsbad, Calif.: 4% Agarose E-gels (Catalog        No. G6018-02);    -   Qiagen, Valencia, Calif.: Proteinase K (Catalog No. 19131); and        Rnase A, DNase-free (Catalog No. 19101).

Additionally, kits and reagents were purchased from the followingvendors: SYBR® Green PCR Master Mix (Applied Biosystems, Foster City,Calif.; Catalog No. 4309155); and QIAamp DNA Mini Kit (Qiagen, Valencia,Calif.; Catalog No. 51304).

The following bacterial strains were obtained from the American TypeCulture Collection (ATCC), Manassas, Va.:

Vibrio harveyi ATCC 25919

Vibrio harveyi ATCC 14126

Vibrio splendidus ATCC 33871

Vibrio anguillarum ATCC 43312

Vibrio alginolyticus ATCC 33839

Vibrio penaeicida ATCC 51842

Vibrio proteolyticus ATCC 53559

Escherichia coli ATCC 25922

Escherichia coli ATCC 11229

Bacillus subtilis ATCC 82

Pseudomonas fluorescens Migula ATCC 700830

Vibrio strains were grown in agar or Marine Broth 2216 (DIFCO, Detroit,Mich., Catalog No. 279110) at 30° C. with or without shaking.

Shrimp DNA samples were obtained from Donald V. Lightner, Department ofVeterinary Science and Microbiology, The University of Arizona, Tucson,Ariz. 85721, USA. These included DNA samples from certified disease freeshrimp (SPF) and infected shrimp containing Penaeus monodon-typebaculoviruses (MBV), Taura syndrome virus (TSV), white spot syndromevirus (WSSV), yellow head virus of P. monodon (YHV), InfectiousHypodermal and Hematopoietic Necrosis virus (1HHNV) and InfectiousMyonecrosis virus (IMNV). Additionally, DNA samples were obtained fromcommercial cultured shrimp and from bacteria stains purchased from ATCC.These DNA samples were prepared using the Qiagen QIAamp DNA Mini Kitusing the manufacturer's protocols.

Templates and Primers

DNA oligonucleotide sequences for synthesis of the synthetic Vibrioharveyi template were prepared from the Vibrio harveyi bioluminescenceregulatory protein (LuxR) gene (GenBank Accession Number M55260;Showalter et al., J. Bacteriol. 172(6):2946-2954 (1990)), and weresynthesized using standard phosphoramidite chemistry or purchasedcommercially (Sigma Genosys Company, The Woodlands, Tex.). The DNAconcentration and copy number of the synthetic template targets andsamples were measured spectrophotometrically at 260 nm (OD₂₆₀). Thetemplates were diluted to specific copy numbers in purified water andwere used as the positive controls and standards for assayquantification. Table 3 displays the location in the LuxR gene, sequenceidentification, and length of the template target. The sequences ofprimers useful for Vibrio harveyi detection are given as SEQ ID NO:1 and2.

TABLE 3 Template Sequence Location in Vibrio harveyi LuxR Gene TemplateSize (bp) SEQ ID NO: (GenBank M55260) VHLT 80 3 679-758

Isolation of DNA

A QIAamp DNA Mini kit was used to recover DNA from shrimp tissue. TotalDNA was recovered using the kit reagents and the manufacture'sprocedures. Generally, this involved adding 200 μL of kit extractionbuffer (10 mM Tris-HCl buffer, pH 8.5, 10 mM EDTA, 100 mM NaCl, 0.5%sodium dodecyl sulfate, and 250 μg/mL proteinase K) to 20 mg of shrimptissue in a 1.5 mL microcentrifuge tube. The shrimp tissue was takenfrom the gills or legs. Tissues were broken open in the extractionbuffer by grinding with a stick provided by the manufacturer. The tubecontents were then incubated at 56° C. for at least 30 min until thesample was dissolved. The sample was vortexed for 20 sec. Then, 4 μL ofRNase A (100 mg/mL) was added to the tube and the reaction mixture wasincubated for 2 min at room temperature. The kit lysis buffer (200 μL)was added and the tube was incubated at 70° C. for 10 min. Ethanol (200μL) was then added and the solution was vortexed. The solution was thentransferred to a spin column and centrifuged at 8000 rpm for 1 min.Then, 500 μL of washing buffer was added, and the spin column wascentrifuged again at 8000 rpm for 1 min. After placing the spin columnin a clean collection tube, 500 μL of washing buffer was added to thespin column and it was centrifuged at 13,000 rpm for 4 min. Afterplacing the spin column in a clean 1.5 mL microcentrifuge tube, 100 μLof kit elution buffer was added to the spin column. After incubating atroom temperature for 1 min, the tube was centrifuged at 8000 rpm for 1min. The eluate containing the DNA was collected in a 1.5 mLmicrocentrifuge tube. In the recovered material, the DNA purity wasassessed by conventional OD_(280/260) ratio measurements and the DNAquantity was determined from the OD₂₆₀ measurements. In some cases,samples were diluted with DNase free water for testing.

Example 1

Demonstration of PCR Amplification of Vibrio harveyi DNA using aSynthetic Target

The purpose of this Example was to demonstrate the detection of theVibrio harveyi synthetic template using PCR amplification with theprimers disclosed herein.

Template standards were prepared by 10-fold serial dilutions of thesynthetic Vibrio harveyi template (described above) in DNase free water.Generally, template concentrations of the standard ranged from 10⁵ to 0copies/5 μL. A master mix was prepared by adding 25 μL of the SYBR®Green PCR Master Mix (Applied Biosystems, Foster City, Calif.; CatalogNo. 4309155) with a volume of primer stock solutions sufficient to givea final concentration of 125 nM of forward and 62.5 nM of reverseprimers, and enough DNase free water to make up a final volume of 45μL/reaction. The master mix was maintained on ice until use.

For each reaction, 5 μL of a template standard was first added to thePCR reaction well and then 45 μL of the master mix was added. Thereactions were then thermal cycled for 40 cycles using a temperatureprogram of 95° C. for 15 sec and 60° C. for 1 min with an initialdenaturing step of 95° C. for 10 min. The amplifications were carriedout in a MicroAmp optical 96-well reaction plate using the ABI PRISM7900 thermal cycler (Applied Biosystems, Foster City, Calif.). Duringeach cycle, PCR product formation was detected by monitoring theincrease in fluorescence arising from the interaction of the SYBR® Greenreporter dye with the DNA amplification products. After completion ofPCR, a dissociation curve (melting curve) was generated over the rangeof 60° C. to 95° C. Data were analyzed using the ABI PRISM 7900 SDSsoftware. In addition, PCR product formation was analyzed by agarose gelelectrophoresis using 4% agarose Egels (Invitrogen Life Technologies,Carlsbad, Calif.; Cat No. G6018-02) and the gel manufacture's protocols.

The results, summarized in Table 4, demonstrate that the appropriatesize amplicon product was produced when the Vibrio harveyi template waspresent. The minimum detectable template level was between 1 and 10copies/rxn. Samples containing no template produced no detectableproduct.

Amplification (CT) and amplicon product formation were, respectively,inversely and directly proportional to the logarithm of the startingtemplate concentration.

TABLE 4 Results of PCR Amplification using a Synthetic Target ForwardReverse Minimum Primer, Primer, Template Product Detectable SEQ ID SEQID SEQ ID Size Template NO: NO: NO: (bp) (copies/rxn) 1 2 3 80 1 to 10

Examples 2 and 3 Detection and Quantification of Vibrio harveyi DNA fromBacterial Cultures

The purpose of these Examples was to demonstrate the detection andquantification of Vibrio harveyi using a PCR assay with the primersdisclosed herein.

In these Examples, serial dilutions of the appropriate synthetictemplate Vibrio harveyi DNA (described above) ranging from 10⁵ to 10⁰copies per reaction were amplified using the conditions stated inExample 1. A standard curve (not shown) was generated using the CTvalues determined from each of the synthetic template concentrations byplotting the CT values, with 95% confidence intervals, against thelogarithm of the initial template copy numbers in the standards. Theslope of this curve (i.e., CT versus log concentration) was then used toestimate the copies of Vibrio harveyi in an unknown sample from theirrespective CT values.

Genomic DNA was isolated from two strains of Vibrio harveyi (ATCC 25919and ATCC 14126). The extracted DNA was serially diluted in purifiedwater and used to provide a series of samples ranging in DNAconcentration from 10 ng/μL to 1 pg/μL of total DNA. Negative controlsincluded a water control containing no template and two DNA shrimpsamples (50 ng/rxn) from two strains of non-infected (SPF) shrimp(Litopenaeus vannamei and Penaeus monodon).

The diluted samples were then amplified using the primers SEQ ID NOs:1and 2, and amplification, master mix, thermal cycling conditions andinstrument stated in Examples 1. The CT value for each diluted DNAsample was then assessed from the PCR amplification reactions. Thecopies of Vibrio harveyi in the samples were then estimated from the CTvalue and the slope of the standard CT versus log template concentrationplot. The PCR products were also analyzed by agarose gelelectrophoresis, as described in Example 1.

The results are summarized in Table 5. In the table, the Vibrio harveyicopy number per reaction is given as the mean of three replicates. Theresults indicate that the primer set produced the correct ampliconproduct size from the Vibrio harveyi DNA and detected DNA in thesamples. The detection limits ranged from about 2 copies/rxn to about 25copies/rxn of the Vibrio harveyi genome. No amplification products weredetected in the water control sample or the SPF shrimp samples.

TABLE 5 Results of Detection of DNA from Vibrio Harveyi Strains V.harveyi genome ATCC DNA/rxn V. harveyi Example No. (ng) CT copies/rxn 255919 10 17.6 251838 1 21.2 19051 0.1 25.2 2109 0.01 28.9 193 0.001 32.125 None 0 >40 0 (water) 0 >40 0 (SPF L. vannamei (50 ng)) 0 >40 0 (SPFL. monodon (50 ng)) 3 14126 10 22.6 1097 1 24.9 2406 0.1 29.5 136 0.0132.8 16 0.001 36.6 2 None 0 >40 0 (water) 0 >40 0 (SPF L. vannamei (50ng)) 0 >40 0 (SPF L. monodon (50 ng))

Example 4 Specificity of Vibrio harvevi Primers

The purpose of this Example was to demonstrate that the primersdisclosed herein do not amplify DNA isolated from other bacteria or DNAand RNA of shrimp infected with other shrimp pathogens.

DNA and RNA samples isolated from shrimp infected with MBV, WSSV, YHV,TSV, 1HHNV, IMNV and SPF were tested using the primers and PCR methoddescribed in Examples 2 and 3. In addition, DNA samples isolated fromother Vibrio strains (Vibrio splendidus, Vibrio anguillarum, Vibrioalginolyticus, Vibrio penaeicida and Vibrio proteolyticus) and othernon-Vibrio bacteria strains (Escherichia coli (ATCC 25922), Escherichiacoli (ATCC 11229), Bacillus subtilis (ATCC 82), and Pseudomonasfluorescens Migula (ATCC 700830)) were tested using the primers and PCRmethod described in Examples 2 and 3.

No PCR amplification was observed when testing shrimp DNA or RNA samplesfrom shrimp infected with the various shrimp pathogens nor when testingthe other Vibrio species or non-Vibrio bacterial strains. These findingstaken together demonstrate that the Vibrio harveyi PCR primers andmethods disclosed herein are selective for Vibrio harveyi and supportsthe conclusions that the primers do not react with shrimp DNA, or DNAand RNA from shrimp viruses, other Vibrio species, or other bacteria.

Example 5 Detection of Vibrio harvevi with an Internal Sample ControlUsing PCR

The purpose of this Example was to demonstrate that the Vibrio harveyiprimers disclosed herein can be used in combination with internal samplecontrol (ISC) primers to produce an ISC product in addition to theVibrio harveyi product. The results presented below demonstrate that theISC primers independently amplify sample DNA and do not interfere withthe amplification of Vibrio harveyi DNA. The presence of the ISC productprovides a marker that can be used as an indication that sample DNA ofsufficient quantity and quality had been recovered for the sample fortesting.

ISC primers were derived from the Penaeus monodon actin 1 gene sequence(GenBank: AF100986). In order to promote preferential amplification ofthe Vibrio harveyi amplicon, the ISC primers were designed to amplify aDNA fragment that was larger than the target Vibrio harveyi testamplicons. The ISC primer sequences are given as SEQ ID NOs:4 and 5, andSEQ ID NOs:6 and 7 (see Table 2).

Samples containing Vibrio harveyi and shrimp actin DNA were prepared by10-fold serial dilutions of a genomic DNA preparation obtained from abacterial culture of Vibrio harveyi (ATCC 25919). The DNA content of thesamples ranged from 0.1 ng to 0.1 pg per reaction. Genomic Penaeusmonodon shrimp DNA (10 ng/rxn) from a non-infected shrimp was then addedto each Vibrio harveyi sample and to negative control samples containingno Vibrio harveyi DNA.

A master PCR mix was prepared by combining 15 μL/reaction of the SYBR®Green PCR Master Mix (Applied Biosystems, Foster City, Calif.; CatalogNo. 4309155) with a volume of primer stock solutions (20 μM for each ofthe Vibrio harveyi primers and 10 μM for each of the actin primers)sufficient to give a final concentration of 125 nM for each of theVibrio harveyi forward and reverse primers (SEQ ID NOs:1 and 2,respectively) and 32 nM for each of the actin forward (ActinF3) andreverse primers (ActinR3), SEQ ID NOs:6 and 7, respectively. DNase freewater was added to make up a final volume of 25 μL/reaction. The mastermix was maintained on ice until use.

For each reaction, 5 μL of the samples was first added to the PCRreaction wells and then 25 μL of the master mix was added. The reactionswere then thermal cycled for 40 cycles using a temperature program of95° C. for 15 sec and 60° C. for 1 min with an initial denaturing stepof 95° C. for 5 min. Amplification was carried out in a MicroAmp optical96-well reaction plate using the ABI PRISM 7900 thermal cycler (AppliedBiosystems, Foster City, Calif.).

During each cycle, product formation was monitored by the CT valuedetermined from the increase in fluorescence arising from theinteraction of the SYBR® Green reporter dye with the DNA amplificationproducts, as described above. After 40 cycles a dissociation curve(melting curve) was generated over the range of 60° C. to 95° C. Datawere analyzed using the ABI PRISM 7900 SDS software. In addition, PCRproduct formation was analyzed by agarose gel electrophoresis using 4%agarose Egels (Invitrogen Life Technologies, Carlsbad, Calif.; Cat No.G6018-02) and the gel manufacture's protocols.

The results obtained using ISC primers ActinF3 (SEQ ID NO:6) and ActinR3(SEQ ID NO:7) are shown in FIGS. 1A and 1B. These figures demonstratethe simultaneous amplification of both Vibrio harveyi and controltemplate targets. The specific Vibrio harveyi DNA produced an 80 byproduct with a melting temperature of 77° C. The actin ISC produced a249 by product (Tm=83.4° C.). The Vibrio harveyi product and actininternal control products were detected by both melting curve analysis(FIG. 1A) and gel electrophoresis (FIG. 1B) based on these size andmelting temperature differences. In both the absence of Vibrio harveyitarget and at various Vibrio harveyi DNA target concentrations, the ISCproduct was detected as a single melting-temperature peak at 83.4° C.(as shown in FIG. 1A) and by electrophoresis (as shown in FIG. 1B). Inall samples containing the Vibrio harveyi template, the specific Vibrioharveyi amplicon was detected by both melting temperature (Tm=77° C.)and by gel electrophoresis. These results demonstrate that the actin ISCtemplate co-amplifies with the Vibrio harveyi template and that the PCRamplification and limit of detection of the PCR assay (1 pg Vibrioharveyi DNA) are unaffected by the presence of the ISC.

Examples 6 and 7 Real-Time Detection of Vibrio harveyi usingFluorescently Labeled Probes

These Examples demonstrate that the Vibrio harveyi primers disclosedherein can be used with fluorescently labeled probes for real timedetection of Vibrio harveyi.

Gene sequences for construction of the fluorescently labeled probes wereselected by analysis of the Vibrio harveyi genes and test ampliconsusing Primer Express® v2.0 software, purchased from Applied BioSystemsInc. (Foster City, Calif. 94404). The probe sequences were chosen tofall within the proximal ends of the specific Vibrio harveyi testamplicons and were 20 to 110 bases in length, depending on the size andsequence of the amplicon. Preference for the probe sequences was givento regions with G/C content of 30% to 80% and with higher C than Gcontent, and with no 5′ G. Generally, probe sequences were selectedhaving a Tm of 8 to 10° C. above the respective Tm of the test primers.Probes sequences which cross-hybridized to other species were notselected for use. The probe sequences selected to meet these criteriaare listed in Table 6.

For real-time detection, the probe sequences were dual labeled. Twodifferent labeling approaches were employed. The 5′ end of the probeswere labeled with a fluorophore (6FAM™, Applied Biosystems). The 3′ endwas labeled either with a quencher dye or in the case of minor grovebinding (MGB) probe, the 3′ end was labeled with a quencher dye and aminor grove binder complex. The labeled probes were prepared andpurchased commercially from Applied BioSystems.

TABLE 6 Vibrio harvevi Probe Sequences SEQ ID GenBank 5′ 3′ Probe NO:No: Location Label Label(s) VHLPM 8 M55260 703-729 FAM¹ MGB² VHLPT 9M55260 703-729 FAM TAMRA³ ¹FAM is 6FAM ™ reagent, Applied Biosystems²MGB is MGB ™ Applied Biosystems ³TAMRA is 6-carboxytetramethylrhodamine

Template standards were prepared by 10-fold serial dilutions of thesynthetic Vibrio harveyi templates (SEQ ID NO:3) in DNase free water.Generally, template concentrations of the standards ranged from 10⁷ to 0copies/μL. A master mix was prepared by combining 25 μL/reaction of theTaqMan® Universal Master Mix (Applied Biosystems, Foster City, Calif.;Catalog No. 4326708) with a volume of primer stock solutions (20 μM foreach of the Vibrio harveyi primers) sufficient to give a finalconcentration of 250 nM for each of the appropriate Vibrio harveyiforward and reverse primers, as shown in Table 7, a volume of probestock solution to give a final concentration of 100 nM, and enough DNasefree water to make up a final volume of 45 μL/reaction. The master mixwas maintained on ice until use.

For each reaction, 5 μL of template standard and then 45 μL of themaster mix were added to each PCR reaction well. The reactions were thenthermal cycled for 40 cycles using a temperature program of 95° C. for15 sec and 60° C. for 1 min with an initial denaturing step of 95° C.for 5 min. The amplifications were carried out in a MicroAmp optical96-well reaction plate using a commercial thermal cycler (IdahoTechnologies Inc., Salt Lake, Utah or Applied Biosystems, Foster City,Calif.). During each cycle, PCR product formation was detected bymonitoring the change in fluorescence arising from the fluorescentlylabeled probe.

Data were analyzed using the thermal cycler's software. In addition, PCRproduct formation was analyzed by agarose gel electrophoresis using 4%agarose Egels (Invitrogen Life Technologies, Carlsbad, Calif.; Cat No.G6018-02) using the manufacture's protocols.

The results, summarized in Table 7, demonstrate that the appropriatesize amplicon product was produced for each primer/probe set when theappropriate Vibrio harveyi template was present. The minimum detectabletemplate level was between 100 and 5,000 copies/rxn, depending on theprimers and probe used. Samples containing no template produced nodetectable product.

Amplification (CT) and amplicon product formation were, respectively,inversely and directly proportional to the logarithm of the startingtemplate concentration.

TABLE 7 Results of PCR Amplification Using a Synthetic Target ForwardReverse Minimum Primer, Primer, Template Probe Product Detectable Exam-SEQ ID SEQ ID SEQ ID SEQ ID Size Template ple NO: NO: NO: NO: (bp)(copies/rxn) 6 1 2 3 8 80 5000 7 1 2 3 9 80 100

1. A method for detecting the presence of Vibrio harveyi in a samplecomprising: (i) providing DNA from a sample suspected of containingVibrio harveyi; and (ii) probing the DNA with a probe derived from theisolated Vibrio harveyi diagnostic primer sequence as set forth in SEQID NO:1 or SEQ ID NO:2 under suitable hybridization conditions; whereinthe identification of a hybridizable nucleic acid fragment confirms thepresence of Vibrio harveyi.
 2. The method for detecting the presence ofVibrio harveyi in a sample according to claim 1 wherein the probederived from the isolated Vibrio harveyi diagnostic primer sequence isselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, and the complete complementary sequences thereof.
 3. The methodaccording to claim 1 wherein the probe contains a replication inhibitingmoiety at the 3′ end.
 4. The method according to claim 3 wherein thereplication inhibiting moiety is selected from the group consisting ofdideoxynucleotides, 3′ deoxynucleotides, a sequence of mismatchednucleosides or nucleotides, 3′ phosphate groups and chemical agents. 5.The method according to claim 4 where in the 3′ deoxynucleotide iscordycepin.
 6. The method according to claim 1 wherein the methodfurther comprises the steps of: a) treating an environment from whichthe sample was obtained with a chemical treatment to kill or control theVibrio harveyi; and b) repeating the steps of the method of claim 1 onanother sample taken from the environment in order to assess Vibrioharveyi inactivation by the chemical treatment.
 7. The method accordingto claim 1 wherein the method further comprises the step of: treating anenvironment from which the sample was obtained with a chemical treatmentto kill or control the Vibrio harveyi in order to improve health andgrow-out of shrimp.